T ARGETING THE STROMA IN PDAC BY REPROGRAMMING CAF S

Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer, due to the inefficient current therapeutic strategies. PDAC is characterized by a rich desmoplastic stroma mainly composed by a heterogeneous population of cancer-associated fibroblasts (CAFs).

CAFs contribute to promote cancer progression, immune-suppression and to treatment resistance (222). Targeting the stroma has gained interest with the aim to enhance drug delivery or inhibit its role in cancer cell chemoresistance or immune-suppression.

In contrast to the previous concept that pancreatic tumor stroma is solely tumor promoting (246), certain components, such as myofibroblasts can function in tumor suppression (152). However, this property may be highly dependent on tumor stage, tissue context and composition of the microenvironment. Indeed, elimination of proliferating SMA myofibroblasts resulted in more aggressive tumors (170). Similarly, reduction of fibrotic stroma by genetic inhibition of stroma related Hedgehog pathway promoted tumor progression (51). On the contrary, blockade of stroma derived soluble factors (87), as well as Vitamin D mediated reprogramming of CAFs resulted in decreased tumor volume and increased chemotherapy efficacy. Interestingly, other studies have shown that normal fibroblasts have oncogenic suppressive potential (247, 248).

Inhibitory property of normal tissue fibroblasts on tumor growth was reported earlier in various organs via governing epithelial homeostasis and proliferative quiescence (149, 150). This suggests that, normal fibroblasts could act as tumor suppressors, a function that is lost upon reprogramming to become CAFs.

Thus, we hypothesized that reprogramming of CAFs to become phenotypically closer to normal pancreatic fibroblast (NPF) characteristics, will retain cancer promotion and will not have the negative effects of stroma elimination.

5.1.1 Isolation and gene expression profiling of CAFs

To confirm our hypothesis, we isolated CAFs and NPFs by two different methods, by outgrowth and by cell sorting, and compared their expression profile in order to better understand their role in tumor development. Differential expression study identified 117 commonly upregulated genes in the two CAF populations. Nevertheless, among the most upregulated genes we found several of them belonged to the acute-phase response proteins, where Serum amyloid A3 presented the highest expression levels. Moreover,

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GSEA analysis displayed numerous pathways shared by these two CAF populations including Complement Cascade as the most significantly upregulated one, as well as Cytokine-Receptor Interaction and Innate Immune Response related pathways, such as NFB. However, it has to be taken into consideration that CAFs isolated by outgrowth method do not represent a pure fibroblast population and may undergo gene expression alterations affected by culture conditions. This can explain the discrepancies between the differential expression analysis of CAFs isolated by outgrowth vs cell sorting.

Hence, we decided to further characterize PDGFRα+ subpopulation of CAFs to avoid contamination by other cell types and to study a more physiological scenario of freshly isolated CAFs.

5.1.2 PDGFRα+ CAFs are protumorigenic

A CAF subpopulation expressing PDGFRα is thought to mediate an inflammatory response (140). However, their putative pro-tumorigenic activity has not been properly documented. In this study, we show that PDGFRα+ CAFs possess pro-tumorigenic properties in vivo based on their ability to promote growth of co-injected pancreatic tumor cells in immunocompromised mice. This property is specific for PDGFRα+ fibroblasts isolated from PDAC tumors since the corresponding PDGFRα+ fibroblasts isolated from normal pancreata inhibited tumor growth (Fig 38).

Recent studies have described distinct populations of CAFs (127, 130, 162). A subpopulation, designated as myCAFs, is characterized by elevated expression of αSMA and appear to localize immediately adjacent to the neoplastic cells. A distinct subpopulation, iCAFs, located more distantly from the neoplastic cells and express low levels of αSMA. Instead, these cells display higher levels of secreted IL-6 as well as of other inflammatory mediators (162).

The CAFs isolated in our study, based on the expression of PDGFRα, also have high levels of IL-6, suggesting that they may represent iCAFs. Other similarities between these iCAFs and the PDGFRα+ CAFs isolated here include significant upregulation of cytokine/chemokine-receptor signaling pathways, as well as JAK-STAT signaling.

However, the PDGFRα+ CAFs characterized in this study display significant overexpression of innate immune response-related signaling and high enrichment in cell-to-cell junction pathways, two properties not reported for iCAFs. Thus, it is plausible that the PDGFRα+CAFs described here might represent, as yet another subpopulation of inflammatory CAFs.

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We selected and validated candidate genes that fulfilled our criteria in PDGFRα+

CAFs and NPFs. Among those, druggability, significant overexpression in CAFs but no or low expression in NPFs; functional relevance in PDAC development, as well as in human disease were the more important for selection. Validation of overexpression of candidate genes in CAFs by qPCR narrowed down our list, which finally resulted in the selection of the following genes: Saa3, Has1, Lumican, Haptoglobin and Mesothelin. For all of these genes, overexpression has been reported in cancer (175, 227, 229). Some of them were found expressed in tumor cells as well (227, 249). Saa3 and Hp are acute-phase response inflammatory proteins associated to chronic inflammation (198, 226).

These two genes appeared among the top 25 upregulated genes in inflammatory CAFs of mouse PDAC in a recent study by Ohlund et al (162). Lum and Has1 have important role in fibrosis and ECM remodeling (232), the latter is responsible for producing hyaluronic acid, the matrix component that defines structure and physical properties of the stroma (124). Whereas, Mesothelin is a tumor antigen and is proposed as a reliable marker of pancreatic cancer, its function has not been addressed properly. We hypothesized, that functional studies of these genes would help to better understand to role of CAFs in PDAC development either in immunosuppression or in physically induced therapy resistance.

5.1.3.1 Functional validation of targets by RNAi silencing

shRNA mediated knock down of these targets revealed functional role of the selected target genes in tumor stroma crosstalk. We co-injected tumor cells with CAFs, in which expression of either Saa3, Has1 or Lumican was downregulated, as well as CAFs infected with control shRNA, subcutaneously in the flanks of immunodeficient mice.

Tumor growth monitoring exhibited reduction in tumor size for all the three genes silenced in CAFs compared to CAFs treated with control shRNA. However, the differences were not significant. One explanation could be the low efficiency of the knock down by shRNA. On the other hand, we did not find a candidate for which silencing would enhance pro-tumorigenic activity of CAFs. Further experiment for overexpression of these proteins should be performed in order to ensure the significance of these result.

5.1.3.2 Generation of knockout mouse models by CRISPR in PDAC stroma

To further investigate to role of the selected genes in PDAC development we took advantage of the novel and fast gene editing technology, CRISPR/Cas9, and generated

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knockout mouse models. By this method, it is also possible to induce mutations simultaneously and efficiently ablate genes at the same time.

We injected guide RNA of Has1 into one cell state embryos derived from our

“therapeutic strain”, which resulted in 4 knockout chimeras with the ability to transmit the modification germ line. However, several crosses were utilized in order to finalize a mouse strain for characterization. In addition, multiple mutational events and their identification has to be taken into consideration when using CRISPR/Cas9 system.

Nevertheless, these events occurring through DNA repair mechanism, are not entirely sure to result in knockout mutations. Taken altogether, development of single mutated Has1 knockout mouse model resulted in 44% efficiency.

Next, we challenged the capacity of the system, to generate triple mutant mice to eliminate Lumican, Haptoglobin and Mesothelin at the same time. Indeed, the three sessions of microinjection resulted in 25 chimeras born, from which 3 were single-, 5 double- and 2 triple-mutant mice that were able to transmit the modification to the next generation. However, numerous crosses were necessary to reach the final genotype for characterization. This is due to the problem that NHEJ – mediated gene modifications produced mutations in a highly unpredictable and rather inefficient manner. Therefore, this mouse strain is still under generation, however preliminary data shows normal viability when eliminating these genes. Future studies will address the functional role of these genes in PDAC development.